Abstract Chronic pain represents an immense clinical problem, with over 100 million Americans afflicted and an annual price tag exceeding half a trillion dollars, according to a recent report from the Institute of Medicine. Studies in our lab are designed to identify molecular, cellular, and circuit mechanisms of sensitization in pain pathways with the goal of identifying novel targets for analgesic intervention. Studies performed in our lab previously identified a critical signaling cascade in neurons of the central nucleus of the amygdala (CeA) that underlies central pain sensitization. This pathway is initiated by metabotropic glutamate receptor subtype 5 (mGlu5) activation of extracellular signal-regulated kinase/ERK signaling, leading to increased firing of CeA neurons. This increase in excitability likely contributes to central sensitization associated with persistent pain. Our prior work, and that of several other groups, suggests that neurons in the CeA represent a critical node of neuromodulation underlying the development of chronic pain. An important finding from our prior studies was that this maladaptive plasticity in the CeA leading to persistent pain sensitization is specific to the right hemisphere. That is, no matter the sight of the injury, plasticity in the right (and not left) CeA was responsible for bilateral pain hypersensitivity. Furthermore, manipulation of neural activity only in the right CeA was found to produce bilateral pain sensitization. The mechanisms generating this hemispheric lateralization are completely unknown. In the present application, we will conduct a series of studies aimed at understanding the circuit context of CeA neurons that are activated by acute pain sensitization. We will perform studies aimed at identifying critical inputs, the type of plasticity that occurs at these synapses, and the major outputs of pain-responsive CeA neurons. We will test whether CeA neurons activated in the context of pain sensitization are necessary and sufficient for the development of pain sensitization, ongoing pain and comorbid disorders. By specifically targeting pain- activated neurons in this study, we may be able to determine if they possess unique neurochemical properties that represent novel therapeutic targets, or genetic signatures that would enable future studies to more precisely determine their function. In vivo 2-photon imaging and microendoscope cameras will be used to monitor activity of these neurons using genetically-encoded Ca2+ sensors, over days to weeks, to determine how the properties of these neurons change during the transition from acute to persistent pain. We will ask whether the population of neurons responsive to heat, cold, or touch change over time, and whether altered activity of these neurons in persistent pain conditions can be normalized using treatments that reduce pain or comorbid anxiety. These studies employ a host of modern techniques including advanced viral tracing, genetic mapping, in vivo calcium imaging, and optogenetic approaches, together with technologies developed in our lab for wireless optogenetic studies to address these important questions.